A sulfatase which hydrolyzes an O-sulfate ester in a glucosamine derivative bearing a second sulfate on the amino group has been purified from human urine. The enzyme has a pH optimum of 6.3 and is inhibited by chloride and sulfate anions. No activity has been detected in fibroblast extracts. To establish the specificity of the enzyme and its relationship to physiological substrates, unequivocal synthesis of specifically sulfated alpha-methyl-2-deoxy-2-sulfamino-6-O-sulfate-D-glucose and alpha-methyl-2-deoxy-2-sulfamino-4-O-sulfate-D-glucose have been carried out. It has been established that the 6-O-sulfate, the prevalent structure in heparin, is not a substrate for this enzyme.